SENSORS AND ACTUATORS B-CHEMICAL
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2021
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IF 7.46
Baoqing Zhou,Qinghua Ye,Fan Li,Xinran Xiang,Yuting Shang,Chufang Wang,Yanna Shao,Liang Xue,Jumei Zhang,Juan Wang,Yu Ding,Moutong Chen,Qingping Wu
Rapid, accurate point-of-care testing (POCT) for pathogenic bacteria detection is the key for avoiding foodborne diseases caused by pathogens or their toxins. The lateral flow biosensor (LFB) based on clustered regularly interspaced short palindromic repeats (CRISPR/Cas) has displayed remarkable potential for pathogen diagnosis. In this study, we report a novel CRISPR/Cas12a-based fluorescence enhanced LFB in conjunction with functionalized quantum dots , combined with recombinase-assisted amplification (RAA), to establish low-cost, simple, and sensitive detection of Staphylococcus aureus , namely CRA-LFB (CRISPR/Cas-recombinase-assisted amplification based LFB). The CRA-LFB assay is characterized by Cas12a-mediated trans-cleavage activation induced by the target DNA to digest biotin-DNA probes, caused no complementarity to the capture probe immobilized on the test (T) line, and resulted in an undetectable T line fluorescence signal on LFB. The naked eye or fluorescence strip reader was used to determine the fluorescence intensity of the T and control lines. The limit of detection (at optimal conditions) was as low as 75 aM of genomic DNA, and 5.4 × 10 2 cfu/mL of S. aureus in pure cultures were detected. Moreover, this CRA-LFB assay can rapidly and accurately detect S. aureus in spiked and natural meat and vegetable samples. The CRA-LFB method yielded high specificity and no interference from other nontargeted bacteria. The results exhibited high-resolution, high-intensity fluorescent signals obtained within 70 min. This bacterial detection platform is simple, low cost, almost equipment-free, and fully qualified for food and clinical diagnosis onsite testing requirements.
研究领域:
生物技术
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微生物学
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食品安全
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即时诊断
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DOI
高影响因子
Synthetic and Systems Biotechnology
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2025
·
IF 4.4
Wen Tian,Songcheng Yu,Kaiyang Zhang,Tao Liu,Lihua Ding,Peng Zhang
The CRISPR/Cas12a system holds significant promise for biomedical applications. Nevertheless, the commonly used reporter, fluorophore–quencher-labeled substrates, is hindered by labor-intensive synthesis procedures and high costs, while also relying on a single-photon method and being vulnerable to environmental interference. Herein, a label-free dual-fluorescent functional nucleic acid (DFFNA) was engineered, comprising an aptamer domain for auramine O (AO) recognition and a dSpacer-integrated DNA duplex region for 5,6,7-trimethyl-1,8-naphthyridin-2-amine (ATMND) binding. The fluorescence of AO and ATMND can be enhanced and quenched, respectively, when bound to DFFNAs. The fluorescence intensity ratio between ATMND and AO increased significantly following the cleavage of DFFNAs by activated Cas12a, thus offering a universal, label-free, ratiometric fluorescent reporter for the CRISPR/Cas12a system. To explore the application of the DFFNA-based CRISPR/Cas12a system, a novel biosensor was developed to detect site-specific DNA methylation. It employs a methylation-sensitive restriction enzyme to recognize methylation sites, Cas12a for site-specific DNA identification and signal amplification, and DFFNAs to produce ratiometric fluorescence. The assay demonstrated remarkable specificity and sensitivity, with a limit of detection of 152 pM, due to the high resolution and trans -cleavage activity of Cas12a. The rationally designed and label-free DFFNAs enhance stability, increase flexibility, and reduce cost. The observable color change and smartphone imaging capability facilitate portable, point-of-care testing. Specifically, the biosensor demonstrated excellent specificity by differentiating colorectal cancer patients from healthy individuals. Consequently, this work presents a superior label-free and ratiometric fluorescent reporter for the CRISPR/Cas12a system, which offers a promising strategy for DNA methylation detection in clinical settings.
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高影响因子
SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
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2025
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IF 4.6
Jiaxuan Chen,Kai Shi
Accurate detection of glyphosate (GLY) residues is essential for protecting public health, ensuring food safety, and preventing environmental pollution caused by excessive or improper herbicide application. Based on the asymmetric CRISPR/Cas12a system as a signal amplification technique, with phosphorothioate-modified hairpin G-quadruplex (psHG4) acting as a signal probe, a platform named ACC-GLY is developed for the sensitive and accurate detection of GLY. In the designed ACC-GLY platform, the target GLY specifically binds to the aptamer, and the cascading signal amplification strategy, driven by the DNase activity of a single Cas12a, is initiated by two competitive guide RNAs. Under the influence of Cas12a's DNase activity, the psHG4 probe is cleaved, releasing the psG4 sequence. The released psG4 sequence then binds to Thioflavin T (ThT), forming a complex that generates a fluorescence signal. Under optimal conditions, the detection platform can specifically detect GLY at a concentration as low as 0.3 pM. The detection platform demonstrates significant capability in detecting GLY in tap water and corn samples, highlighting its vast potential for applications in environmental monitoring and food safety. To the best of our knowledge, this is the first study to utilize the CRISPR/Cas12a system for GLY detection.
研究领域:
分析化学
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生物传感器
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环境监测
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食品安全
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分子生物学
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PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
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2021
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IF 10.618
Bin Qiao,Jiakun Xu,Wenhao Yin,Wanmeng Xin,Lixin Ma,Jie Qiao,Yi Liu
Herein, we report a method that combined “aptamer-locker” DNA with CRISPR/Cas12a-based biosensing for sensitive and rapid melamine analysis. Three strategies were harnessed for designing the DNA sensors that were well characterized by circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC) in the absence and presence of melamine. The detection parameters were optimized to achieve good analytic performance. As a result, a limit of detection (LOD) as low as 38 nM was achieved, which is below the threshold (1.0 mg/kg) of allowable melamine in infant milk products. In addition, the sensors show high selectivity for melamine against other analogues such as cyanuric acid , ammeline and ammelide . Moreover, our method was effective for rapid melamine analysis in whole milk samples, with or without sample pretreatment , in less than 20 min. Adopting a commercially available portable fluorimeter, on-site analysis of melamine in milk was accomplished. The strategies demonstrated here can expand to detect other non-nucleic-acid targets by simply replacing the aptamers.
研究领域:
分析化学
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分子生物学
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生物技术
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食品安全
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DOI
PubMed
高影响因子
ANALYST
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2024
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IF 3.6
Chengchen Tan,Xiaolong Yan,Xingchang Lu,Jianxiu Wang,Xinyao Yi
Breast cancer, as a malignant tumor with the highest incidence in the world, poses a great threat to human life and health. Early diagnosis of breast cancer may help enhancing the survival rate. In this work, a colorimetric and fluorescent dual-mode biosensor based on CRISPR-Cas12a system was constructed for the detection of breast cancer biomarker BRCA1. The intact G4 DNA could form DNAzyme in the assistance of K+ and hemin, catalyzed the oxidation of o-phenylenediamine (OPD) under the assistance of hydrogen peroxide (H2O2), and generate the oxidation product 2,3-diaminophenazine (DAP) with obvious absorption and fluorescence peaks. The presence of the target BRCA1 can activate the trans-cleavage activity of CRISPR-Cas12a, cleaving the G4 DNA and inhibiting the catalytic oxidation of OPD. The target BRCA1 was quantitatively determined by detecting both the absorbance and fluorescence intensity of DAP. Compared with single signal analysis, dual-signal output mode provided more reliable detection results. The detection limits were calculated to be 0.615 nM for colorimetric method and 0.289 nM for fluorescence method. The dual-mode biosensor showed good selectivity for BRCA1 and can resist the interference of complex substrates, which has great potential in biomedical detection.
研究领域:
生物医学工程
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分子生物学
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癌症研究
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生物技术
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高影响因子
MICROCHIMICA ACTA
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2025
·Vol. 192
·期 6
·1-12
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IF 5.3
Chen Yafei,Zhang Xuhui,Huang Wanjin,Jin Zhenhuan,Wei Xiaoping,Li Jianping
Atrazine (ATZ) is used extensively, resulting in residues in food and the environment, posing a serious threat to human health. Herein, Cd 0.5 Zn 0.5 S/Ti 3 C 2 photoelectric material was synthesized and immobilized on a FTO electrode as a photoanode. A photoelectrochemical (PEC) aptamer sensor was constructed for the highly sensitive and selective determination of ATZ based on signal amplification via cascade catalysis of CRISPR/Cas12a and G-quadruplex/hemin DNAzyme (G4/hemin DNAzyme). G4/hemin DNAzyme catalyses the oxidation reaction between H 2 O 2 and dopamine (DA) to form polydopamine (PDA) deposit. This process, in turn, inhibits the photocurrent at the photoanode, leading to a decrease in photocurrent. Concurrently, the depletion of DA as an electron donor for the PEC reaction at the photoelectrode further contributes to the decrease in photocurrent. ATZ can hybridize with ATZ aptamer (Apt) in Apt/cDNA to release activation strand (cDNA), which activates the activity of CRISPR/Cas12a and triggers cleavage of G4, causing the cleaving of G4/hemin DNAzyme immobilized on the electrode surface. This process leads to a decrease of G4/hemin DNAzymes amount on the electrode, consequently reducing both the PDA generation and the DA consumption. As a result, the photocurrent is restored. The cascade catalysis of CRISPR/Cas12a and G4/hemin DNAzyme has been demonstrated to result in photocurrent amplification. The photocurrent change was linear with the logarithmic value of ATZ concentration in the range 1.00 × 10 –12 to 1.00 × 10 –5 mol/L. The limit of detection was 3.47 × 10 –13 mol/L. The sensor has been successfully applied to the determination of trace ATZ in environmental and food samples. Graphical abstract
研究领域:
环境科学
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分析化学
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分子生物学
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PubMed
高影响因子
Frontiers in Microbiology
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2025
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IF 4.5
Yayun Jiang,Zongyao Chen,Xiao Liu,Qi Xin,Dengchao Wang,Caixia Ji,Youwei Li,Gang Mai
Methicillin-resistant Staphylococcus aureus (MRSA), a widely drug-resistant bacterium, poses a significant threat to global health. Current culturing and nucleic acid detection methods are time-consuming and require complex instruments, which do not meet the detection needs. Herein, we developed a rapid and visual MRSA detection method (MCFHCR) using ssDNA-functionalized magnetic beads as a trigger chain combined with the trans-cleavage activity of the Cas12a protein and fluorescence signal amplification of the hybridization chain reaction (HCR). MCFHCR is a signal-off platform for the detection of MRSA. In the absence of DNA targets, the trans-cleavage activity of Cas12a is inactivated, allowing HCR to proceed and form long double-stranded DNA, resulting in an increased fluorescent signal. In the presence of the DNA targets, the trans-cleavage activity of CRISPR/Cas12a is activated to cleave the trigger strand, failing HCR and leading to a decrease in the fluorescence signal. Combined with RPA, MCFHCR was completed within 35 min, achieving a limit of detection (LOD) of five copies/μL for mecA DNA and 8 CFU/mL for MRSA. In detecting clinical strains, MCFHCR demonstrated comparable performance to qPCR and drug sensitivity testing. Therefore, with its simple, rapid operation and convenient signal acquisition, MCFHCR shows significant practical applicability in detecting MRSA.
研究领域:
微生物学
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分子诊断学
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CRISPR技术
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传染病检测
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生物传感器
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PubMed
高影响因子
MICROCHEMICAL JOURNAL
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2024
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IF 4.9
Xuan Wu,Ning-Ning Pi,Fei Deng,Shi-Yang Tang,Cheng-Chen Zhang,Lu Zhu,Fang-Fang Sun,Xiao-Yao He,Han-Qing Li,Shi-Long Zhao,Rong Xiang,Yi Li
Periodontitis not only leads to tooth loss but is also associated with various systemic symptoms. Porphyromonas gingivalis ( Pg ) is the microorganism most closely linked to periodontitis. A rapid and accurate Pg detection method holds significant potential health benefits for patients. While several nucleic acid amplification-based diagnostic methods have been developed, there is a current lack of rapid diagnostic tools for Pg , particularly at chair-side level. Here, two detection methods for the Pg 16S gene based on the CRISPR/Cas12a system have been developed. To achieve ultra-high sensitivity in the developed CRISPR/Cas12a assays without pre-amplification, we designed a circular DNA molecule (termed as Cir-amplifier) which amplifies the signal output of the Cas12a reactions in a cascade response manner. The Cir-amplifier enhanced Fluorescence-Cas12a reaction achieved a minimum detectable limit of 0.39 CFU of Pg within 80-minute under room-temperature. In addition, the Cir-amplifier enhanced Lateral Flow Detection (LFD)-Cas12a reaction has demonstrated the ability to detect a minimum of 3.9 CFU of Pg in 90 min, with results observable by the naked eye. The potential of the two methods for Pg detection in clinical samples has been demonstrated, showing 100 % sensitivity compared to the traditional real-time PCR method. In summary, we have established two efficient detection methods for Pg that eliminate the need for pre-amplification, hence no amplicon contaminations, making them promising candidates for chair-side Pg diagnosis. Furthermore, the Cir-amplifier has been demonstrated to be adaptable to CRISPR/Cas systems producing various signal formats. The Cir-amplifier is expected to become a universal CRISPR/Cas enhancer.
研究领域:
分子生物学
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微生物学
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诊断学
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生物技术
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DOI
高影响因子
TALANTA
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2022
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IF 6.556
Biyun Fang,Zhenzhen Jia,Cui Liu,Kangsheng Tu,Mingzhen Zhang,Lei Zhang
Pathogen nucleic acid detection is of great significance to control the spread of diseases caused by the viruses. Nevertheless, traditional methods for nucleic acid detection such as polymerase chain reaction (PCR) and oligonucleotide microarrays require bulky instruments, which restrain their point-of-care (POC) testing application. Here, we proposed a POC method enabling sensitive detection of pathogen nucleic acids by combining the clustered regularly interspaced short palindromic repeat (CRISPR) Cas12a-based assay and personal glucometer readout (PGM). The quantification of target pathogen DNA by PGM was achieved based on pathogen DNA activates Cas12a ssDNase to cleave magnetic bead-DNA-invertase reporter probe, and separated free invertase to catalyze hydrolysis of sucrose to glucose. Without using nucleic acid amplification technology, we demonstrated here dual signal amplifications based on Cas12a and invertase-mediated catalytic reactions, making it possible to sensitively detect HIV-related DNA or SARS-CoV-2 pseudovirus with the limits of detection of 11.0 fM and 50 copies/μL, respectively. This strategy also showed excellent selectivity as well as potential applicability for detection of HIV in human serum samples or of SARS-CoV-2 in saliva samples. Therefore, our CRISPR-PGM-based dual signal amplifications detection platform might offer a great promise in POC diagnosis of pathogen nucleic acids.
研究领域:
分子生物学
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医学诊断
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生物技术
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Kai Shi,Wenjie Luo,Ying Cheng,Honglei Li,Liai Peng,Xiangrui Luo,Yu Hu,Jiaheng Zhang,Jiaxuan Chen
CRISPR/Cas12a-powered biosensors with guanine (G)-rich sequence reporters (e.g., G-quadruplex and G-triplex) are widely used in detection applications due to their simplicity and sensitivity. However, when these biosensors are employed for molecular detection in complex samples, they may encounter difficulties such as high background signal and susceptibility to interference because of the “turn-off” signal output. Herein, we explore, for the first time, a set of phosphorothioate (ps)-modified G-quadruplex (G4) and G-triplex (G3) sequences that can bind with thioflavin T (ThT) in an active split CRISPR/Cas12a system (SCas12a) to generate a “turn-on” fluorescent signal. To apply this new phenomenon, we develop a universal SCas12a-powered biosensor for “turn-on” fluorescent detection of nucleic acid (miRNA-21) and non-nucleic acid (kanamycin) targets by using ps-modified hairpin G3 as a reporter (SCas12a/psHG3). Target recognition activates SCas12a’s trans-cleavage activity, leading to cleavage at the loop region of the psHG3 reporter. The released prelocked psG3 DNA binds ThT to produce a strong fluorescence signal. Without preamplification, this strategy can detect miRNA-21 with a detection limit of 100 fM. Moreover, the SCas12a/psHG3 system was further utilized for detecting kanamycin by incorporating its aptamers, enabling the detection of kanamycin at concentrations as low as 100 pM. This work is the first to develop a “turn-on” SCas12a/psHG3 system, showcasing its improved performance and wide range of applications in synthetic biology-based sensing technology.
研究领域:
生物技术
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分子生物学
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合成生物学
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高影响因子
MICROCHEMICAL JOURNAL
·
2024
·
IF 4.9
Maolin Li,Yuting Zhang,Xiuyuan Jiang,Jiao Jiang,Xina Gu,Hua Zhang
Transcription factors play crucial roles in governing gene expression and are implicated in various physiological and pathological processes. Here, we propose a novel electrochemiluminescence (ECL) biosensor for the sensitive and specific detection of the transcription factor NF-kB p50, utilizing CRISPR/Cas12a-assisted hyperbranched rolling circle amplification (HRCA) technology. The biosensor achieves a remarkable limit of detection (LOD) of 3.6 fM and demonstrates excellent specificity against interfering proteins. Optimization of experimental parameters further enhances the biosensor’s performance, ensuring its reliability and accuracy. In addition, validation in real biological samples confirms the feasibility of biosensor for practical applications, offering a promising tool for clinical trials and disease diagnosis.
研究领域:
生物技术
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分子生物学
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分析化学
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临床诊断
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DOI
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2024
·
IF 5.7
Yan Zhao,Qianqian Yu,Miaolin Duan,Qi Zhang,Zhishang Li,Yan Zhang,Yana Liu,Han Wang,Xingmin Li,Ruitong Dai,Fei Jia
The efficient, sensitive, and rapid detection of Salmonella typhimurium (S. typhimurium) in food and food products is important to ensure food safety and health. This study developed a fluorescence biosensing assay that integrated recombinase-aided amplification (RAA) and CRISPR/Cas12a with a zeolitic imidazolate framework-8@fluorescein sodium (ZIF-8@FLS) nanocomposite for the sensitive detection of S. typhimurium. In this approach, using RAA as a preamplification module, CRISPR/Cas12a-AChE as a target recognition and dual-enzyme cascade amplification module, and the prepared ZIF-8@FLS with high porosity and rapid pH responsiveness as a fluorescence signal explosive amplification module, the RAA-CRISPR/Cas12a-ZIF-8@FLS biosensor was constructed. Under optimal conditions, it exhibited an excellent linear relationship for S. Typhimurium, with a sensitive detection limit as low as 1.3 × 102 CFU/mL and could complete sample detection within 2 h relying on the RAA and ZIF-8@FLS explosive fluorescence rapid response, demonstrating its significant advantages in specificity, sensitivity, and reliability in food-borne pathogens detection.
研究领域:
食品科学
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生物传感器
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分子生物学
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分析化学
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PubMed
高影响因子
危险品化学品经营许可证（带存储）